JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS
`Volume 26, Number 3, 2010
`© Mary Ann Liebert, Inc.
`DOI: 10.1089/jop.2010.0003
`
`In Vitro Toxicity of Topical Ocular Prostaglandin Analogs
`and Preservatives on Corneal Epithelial Cells
`
`Malik Y. Kahook and David A. Ammar
`
`Abstract
`
`Purpose: To determine the effect of 4 formulations of commercially available prostaglandin analogs (PGAs) on
`human corneal epithelial cells in vitro.
`Methods: The test solutions (PGAs) examined were tafluprost 0.005% with 0.010% benzalkonium chloride
`(BAK), travoprost 0.004% with 0.015% BAK, travoprost 0.004% with soniaTM, and latanoprost 0.005% with
`0.020% BAK. Also tested independently were the 4 respective BAK or sonia concentrations related to each PGA.
`Balanced salt solution (B88) was used as the live control, and a fixative solution containing 70% methanol and
`0.2% saponin was used as the dead control. Immortalized human corneal epithelial cells were exposed to test or
`control solution for 25 min at 370C and 5% C02. A live / dead assay was used to measure the toxicity of the PGAs.
`Results: The percentage of live cells in the PGA groups ranged from 2% to 72% of the B55 group (live control).
`The PGA with the highest relative live cell percentage, at 72% of the live control, was travoprost with sonia. The
`next highest PGA, exhibiting 14% live cells, was the formulation of travoprost containing BAK. The other 2
`PGAs, tafluprost and latanoprost, had few surviving cells, with 3% and 2% live cells, respectively. The BAK
`concentrations exhibited 4%, 3%, and 3% for the 0.01%, 0.015%, and 0.02% concentrations, respectively. The
`stand—alone sonia cell survival was 68% of the live control.
`
`Conclusions: A114 PGA formulations tested demonstrated significantly more toxicity in human corneal epithelial
`cells than the live control, but there were significant differences among the PGAs. Travoprost with sonia
`exhibited the least toxicity, followed by travoprost with BAK, and then tafluprost and latanoprost. The stand-
`alone preservative systems were also tested and showed similar survival percentages to each respective PGA.
`The true clinical implications of these findings require further investigation.
`
`Introduction
`
`USE OF TOPICAL MEDICATIONS to decrease intraocular
`pressure (101’)
`remains the cornerstone of
`treating
`glaucoma in the United States and across the world.1 Since
`glaucoma is a chronic condition requiring long-term therapy,
`potential deleterious effects to the ocular surface from re—
`peated exposures to topical hypotensive agents are of great
`importance to those treating or being treated for this disease.
`Additionally, more information is needed to better under-
`stand the relative toxicity caused by active ingredients used
`to treat glaucoma compared to the preservative systems
`that are used to protect multidose bottles from pathogen
`contamination.
`
`glaucoma or ocular hypertension, reducing IOP by ~25%—
`30%.2 However, conventionally preserved PGAs are known
`to cause a number of in viva ocular surface alterations, in-
`cluding loss of corneal epithelial tight junctions,3 reduction
`in superficial epithelial density,4 and an increase in basal
`epithelial density.5 In vitro studies have also shown that
`PGAs produce expression of inflammatory markers on the
`surface of conjunctival cells5 as well as induce toxicity in
`ocular surface cell lines.6'8
`
`The aim of this study was to compare the relative effects of
`4 PGA formulations with varying concentrations of ben-
`zalkonium chloride (BAK) or the oxidizing preservative
`soniaTM on cultured human corneal epithelial cells. Critical
`to this study was the comparison between 2 formulations of
`travoprost (with and without BAK) that could shed light
`One of the most commonly prescribed classes of hypo-
`on the relative contributions to cell toxicity of the active in-
`tensive agents are prostaglandin analogs (PGAs), which are
`gredient and the preservative systems.
`frequently used as first-line monotherapy for patients with
`
`
`Department of Ophthalmology, Rocky Mountain Lions Eye Institute, University of Colorado Denver School of Medicine, Aurora,
`Colorado.
`
`259
`
`Argentum Pharm. LLC v. Alcon Research, Ltd.
`Case IPR2017-01053
`
`ALCON 2136
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`260
`
`Methods
`
`Cell culture
`
`The transformed human corneal epithelial cell line (10.014
`pRSV-T) was obtained from the American Type Culture
`Collection (Manassas, VA). Corneal epithelial cells were
`cultured at 378C and 5% CO2 in Keratinocyte-Serum-Free
`Medium (Invitrogen, Carlsbad, CA) containing 5 ng/mL
`human recombinant epidermal growth factor (Invitrogen),
`0.05 mg/mL bovine pituitary extract (Invitrogen), 0.005 mg/
`mL insulin (Sigma-Aldrich Corp., St. Louis, MO), 500 ng/mL
`hydrocortisone
`(Sigma-Aldrich Corp.), and antibiotics.
`Flasks and plates used for culturing were previously coated
`with 0.01 mg/mL bovine serum albumin (Sigma-Aldrich
`Corp.), 0.01 mg/mL human fibronectin (BD Biosciences, San
`Jose, CA), and 0.03 mg/mL bovine collagen type I (BD
`Biosciences) for 2 h at 378C.
`
`Reagents
`
`The test solutions were tafluprost 0.0015% with 0.01%
`BAK (Taflotan; Santen Pharmaceutical Co., Ltd., Osaka,
`Japan), travoprost 0.004% with 0.015% BAK (Travatan; Alcon
`Laboratories, Inc., Fort Worth, TX), travoprost 0.004% with
`sofZia (Travatan Z; Alcon Laboratories, Inc.), sofZia preser-
`vative alone (Alcon Laboratories, Inc.), latanoprost 0.005%
`with 0.02% BAK (Xalatan; Pfizer, Inc., New York, NY), as
`well as a range of BAK concentrations (Alcon Laboratories,
`Inc.). The live control was balanced salt solution (BSS; Alcon
`Laboratories, Inc.) and contained the following: 6.5 g/L so-
`dium chloride (NaCl), 0.75 g/L potassium chloride (KCl),
`0.48 g/L calcium chloride dihydrate (CaCl2 2H2O), 0.3 g/L
`magnesium chloride hexahydrate (MgCl2 6H2O), 3.9 g/L
`sodium acetate trihydrate (C2H3NaO2 3H2O), 1.7 g/L sodium
`citrate dihydrate (C6H5Na3O7 2H2O), and sodium hydroxide
`and/or hydrochloric acid to adjust pH to *7.5. The dead
`control was a fixative solution containing 70% methanol and
`0.2% saponin in phosphate-buffered saline (PBS).
`LIVE/DEADÒ Viability/Cytotoxicity Kit for mammalian
`cells (Invitrogen) contained stock solutions of 2 mM ethidium
`
`KAHOOK AND AMMAR
`
`homodimer (Eth-1) and 4 mM Calcein-AM dissolved in di-
`methyl sulfoxide (DMSO). Dulbecco’s PBS (D-PBS) without
`calcium or magnesium (Invitrogen) was used to prepare all
`stains before use. D-PBS had an approximate pH of 7.4 and
`contained the following: 0.2 g/L KCl, 0.2 g/L potassium
`phosphate monobasic (KH2PO4), 8 g/L NaCl, and 2.16 g/L
`sodium phosphate dibasic heptahydrate (Na2HPO4 7H2O).
`
`Experimental procedure
`
`Fifty thousand human corneal epithelial cells were plated
`into each well of a coated 96-well plate in the culture me-
`dium. Cells were assayed upon reaching confluence, usually
`2–3 days postplating. The culture medium was removed by
`aspiration and replaced with 100 mL of test or control solu-
`tion (each solution was performed in triplicate). Cells were
`then incubated at 378C and 5% CO2 for 25 min. After incu-
`bation,
`test solutions were removed and replaced with
`100 mL of D-PBS containing 2 mM Calcein-AM. The final
`concentration of DMSO from the stain stock solution was
`0.1%, a level generally innocuous to most cells.
`Fluorescence was quantified within 20 min of addition of
`stain in a SynergyÔ 4 Multi-Mode Microplate Reader using
`the Gen5Ô Reader Control and Data Analysis Software
`(BioTek, Winooski, VT). Live cells were quantified by de-
`termining the Calcein fluorescence emission at 528 20 nm
`from a 485 20 nm excitation using band-pass filters (F528).
`
`Determination of live cells
`
`Fluorescent data were analyzed as outlined in the manu-
`facturer’s instructions. Briefly, all F528 fluorescence from cells
`stained with 2 mM Calcein-AM was corrected by first sub-
`tracting the F528 fluorescence of cells in wells lacking Calcein
`stain, as this represents the nonspecific fluorescence. The
`percent of live cells in each well was then determined by
`dividing the corrected F528 fluorescence by the average cor-
`rected F528 fluorescence in the BSS-treated cells from each
`experiment (100% Live Control). Data for each treatment are
`reported as mean standard deviation (n¼ 9).
`
`FIG. 1. Live cell assay. The percent of live corneal epithelial cells after a 25 min exposure to 4 different topical ocular
`prostaglandin analogs and control solutions is shown. The number of live cells was normalized to the number of live cells in
`balanced salt solution (BSS)–treated controls. Data are reported as the mean standard deviation of n¼ 9 replicates.
`
`

`

`PROSTAGLANDIN ANALOG EFFECTS ON CORNEAL CELLS
`
`261
`
`Imaging of living/dead epithelial cells
`
`An inverted IX81 microscope (Olympus, Center Valley,
`PA) with spinning-disk using filters sets for fluorescein iso-
`thiocyanate (FITC; excitation 480 20 nm, emission 535
`25 nm) and tetramethylrhodamine isothiocyanate (TRITC;
`excitation 535 25 nm, emission 610 37.5 nm) was used to
`image live (FITC) and dead (TRITC) cells. Cells were viewed
`using a long working distance PLAN Fluorite 40 objective,
`and images were taken with a Hamamatsu ORCA IIER
`monochromatic CCD camera using Intelligent Imaging Sli-
`debook acquisition software (Olympus).
`
`Statistical analysis
`
`Each experiment was performed in triplicate. Three inde-
`pendent experiments were performed on 3 different dates.
`Results represent the means of 9 wells/test condition (3 wells
`each in 3 different assays), expressed as a percentage of BSS-
`treated live control cells. Mean values for each concentration
`were analyzed by the Student’s t-test (Excel, Microsoft,
`Redmond WA); the level of significance was set at 0.05.
`
`Results
`
`As shown in Fig. 1, the percentage of live cells in the test
`solutions relative to BSS control solution ranged from 2% to
`72%, depending on the PGA. The PGA with the highest
`relative live cell percentage was travoprost with sofZia, with
`72% live cells (P < 0.00001). The next highest PGA, exhibiting
`14% live cells, was the formulation of travoprost containing
`BAK. The other 2 PGAs, tafluprost and latanoprost, had few
`surviving cells, with 3% and 2% live cells, respectively. The
`BAK concentrations exhibited 4%, 3%, and 3% for the 0.01%,
`0.015%, and 0.02% concentrations, respectively. The stand-
`alone sofZia cell survival was 68% of the live control, sta-
`tistically identical to travoprost with sofZia (72%, P¼ 23).
`However, travoprost with BAK had statistically fewer live
`cells (14%) than travoprost with sofZia (72%, P < 0.0001). The
`above data is illustrated qualitatively in Figure 2, which
`shows representative images of the corneal epithelial cells
`treated with the various test and control solutions.
`
`Discussion
`
`The current study uses an assay that distinguishes living
`cells by their ability to hydrolyze the cell-permeable (but
`nonfluorescent) Calcein-AM into the nonpermeable but
`strongly fluorescent green Calcein dye. Similarly, the leaky
`plasma and nuclear membranes of dead cells allow the entry
`of the red nuclear stain Eth-1. We used this live/dead cell
`assay to determine the effects of 4 PGA formulations on
`cornea cells in vitro. The respective preservative from each
`medication was also independently evaluated. Latanoprost
`and tafluprost were nearly indistinguishable from dead
`controls, with nearly 100% toxicity of the human corneal
`epithelial cells. Travoprost with sofZia had the highest per-
`centage of live cells, followed by the original BAK-containing
`formulation of travoprost. The preservative systems per-
`formed similarly to their respective PGA. This,
`to our
`knowledge, is the first study to demonstrate the stand-alone
`effects of sofZia on cultured human corneal epithelial cells.
`Our results are consistent with previous publications show-
`ing that travoprost with sofZia produces fewer ocular sur-
`
`face changes than other PGAs, and the current data indicate
`that the preservative systems are the likely culprit for the
`observed differences.3,7,9–14
`BAK is the most common preservative used in topical
`ophthalmic medications and is believed to be a major cause
`
`FIG. 2. Qualitative representation of data presented in
`Fig. 1. Corneal epithelial cells are labeled in green (live) or
`red (dead).
`Increasing concentrations of benzalkonium
`chloride (BAK) increase the number of dead cells. Compared
`to travoprostþ BAK, travoprostþ sofZia results in signifi-
`cantly more live corneal epithelial cells.
`
`

`

`262
`
`KAHOOK AND AMMAR
`
`of toxicity noted with PGA formulations.15–24 BAK is a
`quaternary ammonium compound that acts as a detergent,
`disrupting bacterial cell membranes and ultimately leading
`to bacterial cell death. Similar effects for BAK have been
`noted in numerous in vitro studies on human corneal and
`conjunctival epithelium and stroma.15,21–24 Alternative pre-
`servative systems such as the oxidizing preservative sofZia
`have been developed to potentially diminish the deleterious
`effects on epithelial cells after chronic exposures while still
`protecting multidose bottles from pathogen contamination.
`Oxidizing preservatives cause oxidative damage in bacteria
`and subsequent death due to the lack of oxidases and cata-
`lases in these organisms. Human cells possess these enzymes
`and are thus not similarly harmed.
`This study compares the toxicity of travoprost with 2
`different preservatives, a head-to-head comparison since the
`active ingredient is the same. This study appears to support
`the fact that oxidizing preservatives, in this case sofZia, are
`less toxic than common concentrations of BAK. It is impor-
`tant to emphasize that the clinical significance of the toxicity
`differences observed among the 4 PGA formulations has not
`yet been firmly established. In short-term studies, all 4 PGA
`formulations show little to no adverse effects on the ocular
`surface.25–28 However, since glaucoma is a chronic condition,
`the safety of the long-term use of these agents is of critical
`importance. Clinical studies involving patients on chronic
`topical glaucoma therapy show a link between various oc-
`ular surface disease metrics and increasing number of topical
`drops.29,30 Still, the majority of clinical studies fail to show a
`direct dose-related effect of BAK on ocular surface health
`matching the plethora of available in vitro data.4,5 This is
`likely a result of the poor metrics we currently have available
`to us for in vivo studies. Further studies are needed to better
`understand how in vitro findings correlate with clinical ob-
`servations from once daily dosing with each of these medi-
`cations.
`
`Author Disclosure Statement
`
`Research support was received from Alcon Laboratories,
`Inc., for this study. Dr. Kahook is a consultant for Alcon
`Laboratories, Inc., Allergan, and Merck.
`
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`
`Received: January 5, 2010
`Accepted: March 12, 2010
`
`Address correspondence to:
`Dr. Malik Y. Kahook
`Department of Ophthalmology
`Rocky Mountain Lions Eye Institute
`University of Colorado Denver School of Medicine
`1675 Aurora Court
`PO Box 6510 Mail Stop F-731
`Aurora, CO 80045
`
`E-mail: malik.kahook@gmail.com
`
`

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